Assistant Prof. Dr. Ayad almakki

Department of Clinical Laboratory Science

College of Pharmacy

2 nd stage

Medical microbiology II (Parasitology)

University of Basra

Dr. Ayad almakki

Introduction

Epidemiology

Life cycle

Morphological features

Pathogenesis

Clinical feature

Diagnosis

Treatment

Prevention

Control

Protozoal infection caused by genusPlasmodium that are transmitted tohuman by the bite of infected femaleanopheles mosquitoes

Malaria is the 2nd leading cause of death from infectious diseases inAfrican, after HIV/AIDS.

The word “malaria” come from the Italian mala aria, meaning “badair”. When the term was coined, it was commonly believed thatmalaria was caused by breathing in bad air

There are two phases in the life cycle

1- Sexual cycle , which occurs primarily in mosquitoes

2- Asexual cycle , which occurs in humans (the intermediate hosts)

Two phases :

Exoerythrocytic ( involves infection of live)

Erythrocytic (involves infection of RBCs)

Etiology

Four Plasmodium species are :

1- P. falciparum ( Malignant tertian malaria)2- P. vivax ( Benign tertian malaria)3- P. ovale ( Ovale tertian malaria)4- P. malariae ( Quartan malaria)

Dr. Ayad almakki

3.2 billion people (half the world’s population)live in areas at risk of malaria transmission in106 countries and territories

In 2016, malaria caused an estimated 216 millionclinical episodes, and 445,000 deaths. An estimated91% of deaths in 2016 were in the WHO AfricanRegion.

Dr. Ayad almakki

Dr. Ayad almakki

Individual with sickle cell trait are partially protected against malaria

Their RBCs have too little ATPase activity and cannot produce sufficient energyto support the growth of the parasite.

Sickle cell anemia

Condition that reduce the incidence of malaria

Glucose- 6-phosphate dehydrogenase (G6PD)

Patient with G6PD deficiency may also be protected against malarial infectionto a lesser degree

Duffy antigen Absence of the Duffy blood group determinants (Fya and Fyb) results in a

relative insusceptibility of these individuals to P. vivax infection.

Dr. Ayad almakki

Dr. Ayad almakki

The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infectedfemale Anopheles mosquito inoculates sporozoites into the human host . Sporozoites infectliver cells and mature into schizonts , which rupture and release merozoites . (Of note, in P.vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses byinvading the bloodstream weeks, or even years later.) After this initial replication in the liver(exo-erythrocytic schizogony ), the parasites undergo asexual multiplication in the erythrocytes(erythrocytic schizogony ). Merozoites infect red blood cells . The ring stage trophozoitesmature into schizonts, which rupture releasing merozoites . Some parasites differentiate intosexual erythrocytic stages (gametocytes) . Blood stage parasites are responsible for the clinicalmanifestations of the disease.

The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested byan Anopheles mosquito during a blood meal . The parasites’ multiplication in the mosquito isknown as the sporogonic cycle . While in the mosquito’s stomach, the microgametespenetrate the macrogametes generating zygotes . The zygotes in turn become motile andelongated (ookinetes) which invade the midgut wall of the mosquito where they develop intooocysts . The oocysts grow, rupture, and release sporozoites , which make their way to themosquito’s salivary glands. Inoculation of the sporozoites into a new human host perpetuatesthe malaria life cycle .

Dr. Ayad almakki

Animated life cycle of Plasmodium spp. in the insect vector and human

To view video

press here

Dr. Ayad almakki

Dr. Ayad almakki

The pathogenicity of malaria is related to the erythrocytic infection

As the number of parasites increases the number of erythrocytes is decreased :-

Free in blood stream : -

The debris of the ruptured cells

Parasite (merozoite)

Parasite by produce

Pyrogens stimulate chemoreceptors of the temperature –regulating mechanism of the host to conserve heat

The characteristic chills and fever of a malarial attack

• Rupture of the parasitized cells

• Lysis of non-parasitized cellsNormocytic and normochromic

anemia

• Inside RBCs : Trophozoites feed on hemoglobin( partial metabolism)

forming the malarial pigment darkening (spleen , brain)

Dr. Ayad almakki

Plasmodium falciparum is the species responsible for fatal malaria due to :

• It invades erythrocytes of all ages

• Tendency for more than one parasite to develop in a single erythrocyte

• Invade erythrocyte accumulated and adhere to the lining blood vessels blockage in vital areas ( brain, lungs, and kidneys) Ischemia

Spleen: enlarged (congestion, hemorrhage)

Liver: enlarged (hypertrophy, congestion)

Bone marrow: (congestion, hemorrhage)

Dr. Ayad almakki

Primary attack ( Febrile paroxysm)

• Initial chill is less pronounced and the fever more prolonged

• Sudden uncontrollable shivering followed by

• High grade fever > 40 °C accompanied by headache, muscular pains, nausea and vomiting (N/V) , abdominal pain and increased pulse and respiration rates

• Drenching sweat the patient is exhausted but feels marked relief.

• Pernicious symptoms ( coma, convulsions, cardiac failure) rarely occur .

P. vivax, p. ovale and P. malariae

P. falciparum

• Pernicious manifestations are common

It includes several paroxysms of symptoms of gradually decreasing intensity over a period of 3 weeks:-

Dr. Ayad almakki

Relapse

Several attacks occur after long period of remission ( > one year)

P. vivax and P. ovale due to persistence of exoerythrocysticdevelopment ( dormant parasite)

Recrudescence

Renewal of clinical manifestation after long period of remission without re-exposure

P. Falciparum and P. malariae due to persistence of parasites in the blood at levels too low to be detected or to produce symptoms

Dr. Ayad almakki

3- Gastrointestinal disorders

4- Vascular collapse and shock (Algid malaria)

5- Malarial hyperpyrexia : Temperature > 42 C

2- Black water fever :-

Massive destruction of uninfected RBCs by autoantibodies activated by antigen ( newly parasitized or quinine treated RBC)

Sever chills with rigor, high fever, jaundice, vomiting , rapidly progressive anemia and dark red or black urine ( color of cola)

Complications in malaria

Mainly due to P. Falciparum infection :

1- Cerebral malaria

Congestion and anoxia of brain Hyperpyrexia , coma , convulsions, Paralysis and death

Complication of P. malariae include nephrosis Nephorticsyndrome in children

Dr. Ayad almakki

Dr. Ayad almakki

Clinical feature are suggested but not diagnosis

Laboratory Diagnosis

Microscopic demonstration still the Gold standard in Diagnosis:

Chronic stage at any time

Timing of blood smear: Acute stage early or at peak of paroxysmal attack

Thick and thin film using Giemsa’s stain : Thick blood smears are more sensitive indetecting malaria parasites because the blood is more concentrated allowing for agreater volume of blood to be examined ; however , thick smears are more difficultto read

Thick smear used to screen for presence of parasite

Thin smear is used for species identifcation

Dr. Ayad almakki

Dr. Ayad almakki

P. falciparum infection of RBCs showing Ring form

P. falciparum infection of RBCs showing gametocytes

Dr. Ayad almakki

Quantitative Buffy Coat (QBC)

is a laboratory test to detect infection with malaria or other blood parasites.The blood is taken in a QBC capillary tube which is coated with acridine orange(a fluorescent dye) and centrifuged; the fluorescing parasites can then beobserved under ultraviolet light at the interface between red blood cells andbuffy coat. This test is more sensitive than the conventional thick smear,however it is unreliable for the differential diagnosis of species of parasite

Procedure:

1-Draw samples of blood ( 55 µl) in to the QBC tube by capillary action.

2-Rotate the tubes for 10 seconds to dissolve the contained residues in the blood.

3-Insert a close fitting cylindrical insert or plastic float inside a acridine orange-coated capillary tube.4-Centrifuge the tubes at 12,000 g for 5 minutes.After gentrification blood components and malaria parasites separate based on density, and concentrate in distinct layers

5-Insert the centrifuged QBC Malaria test into the Previewer. Position the tube so the closure end extends over the depressed area of the holder.

Dr. Ayad almakki

Quantitative Buffy Coat (QBC)

Procedure:

(Cont.)

6-The area surrounding the float just beneath the buffy coat was examined under oil immersion. Individual cells within this layer were easily seen by microscopy; the malaria parasites staining green (DNA) and orange (RNA) under blue-violet light.

7-The entire circumference of the tube was examined systematically while moving away from the buffy coat through the erythrocyte layer.

8-Each tube was examined until parasites were detected or for a maximum of 5 minutes.

Dr. Ayad almakki

Appearance of malarial parasitein QBC system under microscopy Dr. Ayad almakki

QBC capillary tube

Antigen detection method

Various test kits are available to detect antigens derived from malariaparasites. Such immunologic (immunochromatographic) tests mostoften use a dipstick or cassette format , and provide results in 2-15minutes. These “ Rapid Diagnostic Tests”

Dr. Ayad almakki

Serology

Serology detects antibiodies against malaria parasites, using either indirectimmunofluorescence (IFA) or enzymes-linked immunosorbent assay (ELISA).

Serology does not detect current infection but rather measures past experience.

Molecular

Parasite nucleic acids are detected using polymerase chain reaction (PCR). Thistechnique is more accurate than microscopy.

Dr. Ayad almakki

DrugsObjectivesClass definition

example

Quinine,Artemisinins,Amodiaquine,

Chloroquine, Lumefantrine, Tetracycline a ,

Atovaquone, Sulphadoxine, Clindamycin a

, Proguanil a

Act on (erythrocytic) stage of the

parasite thereby terminating

clinical illness

Blood

Schizonticidal

drugs

Primaquine, Pyrimethamine, Proguanil,

Tetracycline

Act on primary tissue forms of

plasmodia which initiate the

erythrocytic stage. They block

further development of the

infection

Tissue

schizonticidal

drugs

Primaquine, Artemisinins, Quinine bDestroy sexual forms of the

parasite thereby preventing

transmission of infection to

mosquitoes

Gametocytocidal

drugs

a slow acting , cannot be used alone to avert clinical symptoms b Weakly gametocytocidal

The pharmacology of antimalarials

Dr. Ayad almakki

DrugsObjectivesClass definition

example

Primaquine, Tafenoquine

These act on persistent liver

stages of P. ovale and P. vivax

which cause recurrent illness

Hypnozoitocidal

drugs

Primaquine, Proguanil, Chlorguanil

These act by affecting further

development of gametocytes

into oocytes within the mosquito

thus abating transmission

Sporozontocidal

drugs

The pharmacology of antimalarials

(Cont.)

Dr. Ayad almakki

ChloroquineMechanism of action

1- The parasite digests the host cell’s hemoglobin to

obtain essential amino acids

2- The process releases large amounts of heme, which is

toxic to the parasite

3- To protect itself the parasite ordinarily polymerizes theheme to nontoxic hemozoin, which is sequestered inthe parasite’s food vaculo

4- Cholroquine prevents the polymerization to hemozoin

5- The accumulation of heme results in lysis of both theparasite and the red blood cell.

Dr. Ayad almakki

Chloroquine

Mechanism of action

Dr. Ayad almakki

Artemisinin derivative

3 more semi-synthetic derivatives that are actually more active than artemisinin

itself, it includes :-1- Artesunate 2- Artemether 3- Arteether

Mechanism of action

Endoperoxide bridge

In the acid vacuole of parasite, cleavage of endoperoxidebridge of artemisinin compounds by heme iron

Free radicals generated

Damage parasite membrane by covalently binding to membrane proteins

Death of parasiteDr. Ayad almakki

Quinine A first-line treatment for malaria, and it should be used only when

artemisinins are not available

Mechanism of action Same like chloroquine

Pharmacokinetics

Like Cinchonism is a pathological condition caused by an overdose of quinine :-

Dose dependent toxicity Side effect

Mild form : Tinnitus , slight reversible impairment of hearing Severe form : vertigo, vomiting, abdominal pain

Hypotension : Occur if drug is given too rapidly

Hypoglycemia : I.V. quinine which is due to release of insulin

Black water fever : a fetal condition in which acute haemolytic anaemia is associated with renal failure( dark urine)

Absorption :- Rapidly absorbed both orally & parenterally Metabolism :- liver Excretion :- urine

Dr. Ayad almakki

Dr. Ayad almakki

Chemoprophylaxis

Should be given 1 week before traveling, and continued 4 weeks after leaving

Depends on the area of travel( ie. Chloroquine resistance or not )

RegimenAdult prophylactic doseAntimalarial tablets

Chloroquine resistance high

Started 2-3 weeks before travel and continued until 4 weeks

after250 mg weeklyMefloquine

Started 1 week before and continued until 4 weeks after

travel100 mg dailyOr Doxycycline

From 1-2 days before travelUntil 1 week after return

1 tablet dailyOr Malarone

Chloroquine resistance absent

Started 1 week before and continued until 4 weeks after

travel

300 mg base weeklyChloroquine

100-200 mg dailyand proguanil

Dr. Ayad almakki

Elimination of Mosquito breeding places

National improvements on health and hygiene

Use of Mosquito nets, treated with Pyrithrin

Clothing with sleeves, and long trousers

Use of Mosquito repellents

Treatment of human infections with antimalarial drugs

Dr. Ayad almakki

Dr. Ayad almakki